Benzoylacetylene derivatives

Abstract: A compound of the following formula (I) wherein R¹∼ R⁵ are independently hydrogen; -OR⁷ wherein R⁷ is hydrogen, C₁∼ C₅ alkyl optionally substituted by halogen or phenyl, or -COR⁸ wherein R⁸ is phenyl or C₁∼ C₅ alkyl optionally substituted by phenyl; halogen; C₁∼ C₅ alkyl optionally substituted by halogen; -NR⁹R¹⁰ wherein R⁹ and R¹⁰ are independently hydrogen, phenyl, C₁∼ C₅ alkyl optionally substituted by phenyl, benzoyl, or acetyl; -SOpR¹¹ wherein p is 0, 1 or 2, R¹¹ is C₁∼ C₅ alkyl or phenyl; cyano; or nitro; or when adjacent substituents are taken together, they may represent C₁∼ C₃ oxyalkylene having one or two oxygen atoms, R⁶ is hydrogen; C₁∼ C₅ alkyl; wherein Y¹, Y² and Y³ are independently hydrogen, pyridyl, C₁∼ C₅ alkyl, phenyl or halogen; wherein Z¹, Z² and Z³ are independently hydrogen, C₁∼ C₅ alkyl or halogen; C₃∼ C₁₂ trialkylsilyl; -COR¹² wherein R¹² is C₁∼ C₅ alkyl, phenyl, C₁∼ C₅ alkoxy optionally substituted by phenyl, or -NR¹³R¹⁴ wherein R¹³ and R¹⁴ are independently hydrogen, C₁∼ C₅ alkyl optionally substituted by phenyl, or phenyl optionally substituted by halogen; or -CR¹⁵R¹⁶X wherein R¹⁵ and R¹⁶ are independently hydrogen, phenyl, C₁∼ C₅ alkyl optionally substituted by phenyl, or C₃∼ C₈ cycloalkyl, or when taken together to be C₃∼ C₇ alkylene optionally substituted by C₁∼ C₅ alkyl, X is -OR¹⁷ wherein R¹⁷ is hydrogen, C₁∼ C₅ alkyl optionally substituted by phenyl, or -COR¹⁸ wherein R¹⁸ is phenyl or C₁∼ C₅ alkyl optionally substituted by phenyl, -COR¹⁹ wherein R¹⁹ is hydroxyl, phenyl optionally substituted by C₁∼ C₅ alkyl, C₁∼ C₅ alkyl optionally substituted by phenyl, C₁∼ C₅ alkoxy optionally substituted by phenyl, or -NR²⁰R²¹ wherein R²⁰ and R²¹ are independently hydrogen, C₁∼ C₅ alkyl or phenyl optionally substituted by halogen, -SOpR²² wherein p is 0, 1 or 2, R²² is C₁∼ C₅ alkyl or phenyl, or -NR²³R²⁴ wherein R²³ and R²⁴ are independently hydrogen, phenyl optionally substituted by halogen or C₁∼ C₅ alkyl, or C₁∼ C₅ alkyl optionally substituted by hydroxyl, phenyl, C₁∼ C₅ alkylamino, C₂∼ C₆ dialkylamino or indolyl, C₃∼ C₈ cycloalkyl, pyridyl, -SO₂R²⁷ wherein R²⁷ is phenyl optionally substituted by C₁ ∼ C₃ alkoxy, or -COR²⁵ wherein R²⁵ is C₁∼ C₅ alkyl, phenyl, or C₁∼ C₅ alkoxy optionally substituted by pyridyl, N-methylpyridyl, N-oxopyridyl or phenyl optionally substituted by carboxyl or sodium carboxylate, or when R²³ and R²⁴ are taken together, they are C₃∼ C₆ alkylene optionally containing therein -O-, -NR²⁶- wherein R²⁶ is hydrogen, phenyl or C₁∼ C₅ alkyl, or C₃∼ C₆ alkylene optionally substituted by C₁∼ C₅ alkyl with a proviso that when R⁶ is hydrogen, R² and R³ are not methoxy, or its salt, and a pharmaceutical composition containing at least one of these compounds, which is useful as a tyrosine kinase inhibitor and useful for suppressing the growth of cancer cells.

Description: FIELD OF THE INVENTION This invention relates to benzoylacetylene derivatives and salts thereof, more particularly to benzoylacetylene derivatives and salts thereof having specific protein kinase (referred to as "tyrosine kinase" hereinafter) inhibiting activity. BACKGROUND OF THE INVENTION In chemotherapy for cancer, a lot of therapeutic agents have been put into practical use. However, most of them are not sufficiently effective in their therapeutic action. In addition, growth inhibiting action of them is not limited to cancer cells, and therefore, they often cause serious side-effects, which make them unsatisfactory therapeutic agents. It is well known that tyrosine kinase plays an important role in intercellular signal transduction and cell differentiation or growth. Accordingly, failure of control of tyrosine kinase activity in cells disorders intercellular signal transduction and causes abnormal cell differentiation/growth, which is considered to be directly responsible for the development of various diseases. In particular, it is known that tyrosine kinase is significantly associated with disorderly overgrowth of cancer cells. In fact, overexpression of tyrosine kinase is epidemiologically observed in various carcinomas. On the basis of this finding, it has been proposed that an agent specifically inhibiting tyrosine kinase activity would be an anti-cancer agent having minor side-effects and exerting its therapeutic effect through novel mechanisms. Examples of such agent are Erbstatin, Lavendustin, Herbimycin A, and Genistein which are all derived from microorganisms. Additional example are synthetic compounds such as benzylidenemalonitrile (Japanese Patent Publication Kokai No. 138238/1990; Journal of Medical Chemistry, 32 p2344, 1989; ibid 34 p1896, 1991), α-cyanosuccinamide derivative (Japanese Patent Publication Kokai No. 222153/1988) 3,5-diisopropyl-4-hydroxystyrene derivative (Japanese Patent Publication Kokai No. 39522/1987), 3-5-di-t-butyl-4-hydroxystyrene derivative (Japanese Patent Publication Kokai No. 39523/1987), and Erbstatin analogue (Japanese Patent Publication Kokai No. 277347/1987). Tyrosine kinase inhibitors previously known possess too moderate inhibitory activity to be used as an anti-cancer agent. Accordingly, the purpose of the present invention is to provide a family of novel compounds useful for suppressing the growth of cancer cells, which compounds are easily available, exhibit specific and intensive activity in inhibiting tyrosine kinase of the growth factor receptor, and show negligible side-effects that anti-cancer agents previously known have. SUMMARY OF THE INVENTION It has found that a new class of benzoylacetylene derivatives have an unprecedented intensive activity to inhibit tyrosine kinase and have an antiproliferative activity. According to one aspect of the present invention, there is provided a compound of the following formula (I) wherein R 2 and R 3 are C 1 -C 3 alkoxy; R 6 is phenyl, pyridyl or -CR 15 R 16 X wherein R 15 and R 16 are independently hydrogen or C 1 -C 5 alkyl, optionally substituted by phenyl, or when taken together, they are C 3 -C 7 alkylene, X is hydroxyl or -NR 23 R 24 wherein R 23 and R 24 are independently hydrogen, phenyl optionally substituted by halogen or C 1 -C 5 alkyl, C 1 -C 5 alkyl optionally substituted by hydroxyl, phenyl or indolyl, pyridyl, -SO 2 R 27 wherein R 27 is 3,4-dimethoxyphenyl, or -COR 25 wherein R 25 is C 1 -C 5 alkoxy optionally substituted by phenyl, pyridyl, N-methylpydridyl or N-oxopyridyl, or when R 23 and R 24 are taken together, they are C 3 -C 6 alkylene optionally substituted by C 1 -C 5 alkyl; or its salt, except 2-propyn-1-one, 1-(3,4-dimethoxyphenyl)-3-phenyl. DETAILED EXPLANATION OF THE INVENTION The compounds of the present invention are useful as pharmaceutical compositions for suppressing the growth of cancer cells, therapeutical treatment of arterial sclerosis, inhibiting platelet aggregation, immunodepression and suppressing inflammation. They are shown by the above formula (I). In the general formula (I), halogen includes fluorine, chlorine, bromine and iodine. C 1 ~ C 5 alkyl includes methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl and neopentyl, C 1 ~ C 5 alkoxy includes methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, n-pentyloxy and neopentyloxy. Further, as salts formable from the benzoylacetylene derivatives represented by the above-noted general formula (I), there may be mentioned for example salts of inorganic acids such as carbonates, hydrogencarbonates, hydrochlorides, sulfates and phosphates, or salts of organic acids such as formates, propionates, oxalates, fumarates, maleates, succinates, tartrates, benzoates, phthalates, methansulfonates and 4-toluenesulfonates. When the benzoylacetylene derivatives have an amino or pyridyl group, oxygen or an alkyl group may combine with nitrogen to form an ammonium salt or pyridinium salt. Preferred examples of the compounds according to the invention are shown in Table-1. R 2 , R 3 and R 6 in the above-noted general formula (I) are represented in the Table. Ac, Bu, Bn, Me, Et, Pr and Ph represent acetyl group, butyl group, benzyl group, methyl group, ethyl group, propyl group and phenyl group, respectively, in the Table (the same as in the following Tables). Preparation of the compounds of the present invention The compounds of the present invention can be prepared for example by the following routes: For example, an acetylene derivative represented by the above-noted general formula (III) can be reacted in a suitable solvent, for example, an ether type solvent such as tetrahydrofuran, diethyl ether, a hydrocarbon type solvent such as benzene, toluene, a proton polar solvent such as methanol, ethanol, as well as a non-proton polar solvent such as dimethyl sulfoxide, and dimethyl formamide, with a base, for example, an organo metal compound such as butyllithium and ethylmagnesium bromide, a metal alcoholate such as sodium methoxide, a metal hydride such as sodium hydride and potassium hydride at a temperature between -100°C and +100°C, preferably -80°C and +50°C, for from 5 minutes to 12 hours to prepare a metal acetylide represented by the general formula (IV), which can be then reacted with a benzaldehyde derivative represented by the general formula (II) at a temperature between -100°C and +100°C, preferably -80°C and +50°C, for from 5 minutes to 24 hours, preferably from 30 minutes to 12 hours, to prepare an adduct represented by general formula (V). The compounds of the present invention can be prepared by oxidizing the compound (V) in a suitable solvent, for example, a hydrocarbon type solvent such as benzene and toluene, a polar solvent such as acetone and water and a halogen type solvent such as dichloromethane and chloroform. As oxidants used for the oxidation, there may be mentioned metal oxidants such as manganese dioxide and chromic acid, organic oxidants such as oxalyl chloride-trifluoroacetic anhydride. Further, the compound represented by the general formula (I) can be prepared by hydrogen transfer reaction of the compound (V) with aluminium isopropoxide or zirconium chloride in a carbonyl compound. Furthermore, the compounds of the present invention can be prepared by a route shown in the following scheme: Thus, the compound represented by the general formula (I) can be prepared by reacting a benzoic acid derivative represented by the general formula (VI) with thionyl chloride or phosphorus pentoxide in a hydrocarbon type solvent such as benzene and toluene or a halogen type solvent such as dichloromethane and chloroform to prepare acid halide compounds (VII) and reacting the compounds (VII) with compounds (IV) in an ether type solvent such as tetrahydrofuran and diethyl ether, a hydrocarbon type solvent such as benzene and toluene or a mixed solvent thereof at a temperature between -100°C and +100°C for from 5 minutes to 24 hours. Furthermorey, the compound represented by the general formula (I) can be prepared by reacting compounds (VI) with compounds (VII) in the presence of a catalytic amount of palladium complex and copper salt in a suitable solvent such as tetrahydrofuran and benzene at a temperature between +10°C and +100°C for from 30 minutes to 48 hours. Furthermore, the compounds represented by the general formula (I), wherein R 6 is -CR 15 R 16 NHCOR 25 (R 15 , R 16 and R 25 being as defined above) can be prepared by reacting an acid anhydride or an acid chloride represented by (R 25 CO) 2 O or ClCOR 25 corresponding to the compound (V) [R 6 =-CR 15 R 16 NH 2 (R 15 and R 16 are defined as above)] in the presence of bases such as sodium hydroxide, potassium hydroxide, pyridine and triethyl amine in a solvent such as methylene chloride, tetrahydrofuran, toluene and water or a mixed solvent thereof to prepere compounds (V) [-CR 15 R 16 NHCR 25 (R 15 , R 16 and R 25 defined as above)] and then subjecting said compound (V) to the above-noted oxidation reaction. Furthermore, the compounds represented by the general formula (I), which are a pyridinium salt or an ammonium salt can be prepared in a desired salt form by reacting the corresponding compound having pyridyl group or amide group with the corresponding acid component such as hydrochloric acid, hydrobromic acid or a solution thereof in an organic solvent, fumaric acid or methyl iodide in a suitable solvent such as diethyl ether and chloroform. Tyrosine kinase inhibitor of the present invention Tyrosine kinase inhibitor of the present invention comprises at least one compound selected from the compounds represented by the above-noted general formula (I) and their pharmaceutically acceptable salts as an effective ingredient. The compounds of the present invention are useful as tyrosine kinase inhibitors as described below, and may be used as anti-cancer agent, immunodeppressor, a platelet aggregation inhibitor, an anti-arteriosclerosis or an anti-inflammatory agent based on their activity. A pharmaceutical composition containing a compound of the formula (I), which is useful as a tyrosine kinase inhibitor can be formulated into various dosage forms for oral, enteral or parenteral administration. Specific formulations include tablets, capsules, fine granules, syrup, suppositories, ointments and injections. As carriers for preparing such composition in the form of pharmaceutical formulation, there are used pharmaceutical carriers suitable for oral, enteral or parenteral administration, such as organic or inorganic, and solid or liquid substances which are inactive. Specifically, the carriers include, for example, microcrystalline cellulose, gelatin, lactose, starch, magnesium stearate, talc, vegetable fat and oil, animal fat and oil, gum and polyalkylene glycol. The ratio of the compound (I) to a carrier in a formulation can vary in the range of the ratio of 0.2 to 100% by weight. The pharmaceutical composition of the invention useful as a tyrosine kinase inhibitor may contain other substances effective as a tyrosine kinase inhibitor and other mischible pharmaceuticals. In this case, the compound of the invention may not be a major component of the composition. The compositions of the invention in the form of a pharmaceutical formulation are generally administered in an amount by which desirable effects can be obtained with no side effect. Particular dosages of the formulation should be determined depending on physician's judgement, but it typically ranges from 10 mg to 10 g, and preferably 20 mg to 5 g, per day for an adult. The compound (I) of the present invention may be administered as an active ingredient in an amount of 1 mg to 5 g, and more preferably 3 mg to 1 g, per day for an adult. The following detailed examples are presented by way of illustration of certain specific embodiments of the invention. The examples are representative only and should not be construed as limiting the invention in any respect. [Examples] Synthesis Example 1 To a suspension of diisopropylamine (4.05g, 40.0mmol) and potassium carbonate (6.91g, 50.0mmol) in acetone (15ml) was added propargyl bromide (3.01ml, 40.0mmol) at 5°C with stirring. The reaction mixture was warmed to room temperature over a period of 2 hours, and then it was stirred at room temperature for further 3 hours. Precipitated substances were filtered off, filtrate was concentrated to give a residue, to which water was added, and a product was extracted with dichloromethane (50ml). After the extract was dried over sodium sulfate, the solvent was distilled off under reduced pressure to yield oily propargyl amine (2.78g, yield 50%). NMR spectra are as follows. 1 H NMR (250MHz, CDCl 3 ) δ ppm : 1.10 (d, J = 6.5Hz, 12H), 2.13 (t, J = 2.5Hz, 1H), 3.20 (hep, J = 6.5Hz, 2H), 3.42 (d, J = 2.5Hz, 2H). Synthesis Example 2 A solution of propargyl amine (1.39g, 10.0mmol) obtained in Synthesis Example 1 in tetrahydrofuran (40ml) was cooled to -70°C under a nitrogen atmosphere, to which a solution of 1.56M n-butyl lithium (6.4ml, 10.0mmol) was added dropwise with stirring. After the reaction solution was warmed slowly to 0°C, it was cooled to -70°C, to which a solution of 3,4-dimethoxy benzaldehyde (1.66g, 10.0mmol) in tetrahydrofuran (10ml) was added dropwise. After the reaction solution was warmed to 0°C for 2 hours, the reaction was quenched by the addition of water. After the solvent was distilled off under reacted pressure, the residue was extracted with dichloromethane (40ml×2). After the extracts were dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developer: CHCl 3 /MeOH=20/1) to give the above-noted aminoalcohol (3.00g, yield 98%) as an oily material. NMR spectra are as follows. 1 H NMR (250 MHz, CDCl 3 ) δ ppm : 1.10 (d, J = 6.5Hz, 12H), 2.40 (brs, 1H), 3.20 (hep, J = 6.5Hz, 2H), 3.51 (d, J = 1.7Hz, 2H), 3.89 and 3.9 0 (s, 6H), 5.42 (brs, 1H), 6.85 (d, J = 8.1 Hz, 1H), 7.07 (m, 2H). Synthesis Example 3 A solution of triphenyl phosphine (3.54g, 13.5mmol) and carbon tetrabromide (2.24g, 6.7mmol) in methylene chloride (30ml) was cooled to 0t under a nitrogen atmosphere, to which mesitoaldehyde 1 (0.5g, 3.37mmol) was added with stirring. The reaction mixture was stirred at 0°C for 30 minutes and thereafter warmed to the room temperature and stirred at room temperature overnight. Pentane (120ml) was added, the precipitated substances were distilled off, a residue was washed with pentane, a filtrate was concentrated, and the solvent was distilled off. A residue was purified by a silica gel column chromatography (developer: hexane) to obtain 2 (0.310g, yield 30.2%). Synthesis Example 4 After a solution of 2 (0.1082g, 0.356mmol) in THF (20ml) was cooled to -78°C under a nitrogen atmosphere, n-butyl lithium (hexane solution, 1.66M, 0.43ml) was added. After the reaction mixture was stirred at -78°C for 1 hour, it was warmed to the room temperature for 1 hour and then stirred at the room temperature for further 1 hour. Thereafter, it was cooled to -78°C again, and veratoraldehyde (0.0592g, 0.356mol) was added and warmed to the room temperature for 2 hours and then stirred at the room temperature for one night. An aqueous ammonium chloride solution was added and the mixture extracted with ethyl acetate. After the extract was dried over sodium sulfate, the solvent was distilled off under reduced pressure. A residue was purified by a silica gel column chromatography (developer: hexane:ethyl acetate=5:1) to give 3 (0.049g, yield 4%). Synthesis Example 5 A solution of 2-bromodiphenyl 5 (1g, 4.29mmol) in THF (40ml) was cooled to -78°C under a nitrogen atmosphere, and n-butyllithium (hexane solution, 1.70M, 3ml) was added with stirring. Alter stirring for 30 minutes, DMF (0.47g, 6.44mmol) was added and warmed to the room temperature for 2 hours and stirred for one night. An aqueous solution of ammonium chloride solution was added and the mixture extracted with ethyl ether. After the extract was dried on sodium sulfate, the solvent was distilled off under a decreased pressure. A residue was purified by a silica gel column chromatography (developer: hexane:ethyl acetate=10:1) to obtain 6 (0.7484g, yield 96%). Synthesis Example 6 A solution of triphenyl phosphine (4.3g, 16.4mmol) and carbon tetrabromide (2.72g, 8.2mmol) in methylene chloride (40ml) was cooled to 0°C under a nitrogen atmosphere, and 6 (0.7484g, 4.lmmol) was added with stirring. After the reaction mixture was stirred at 0t for 30 minutes, it was warmed to room temperature and stirred at room temperature over night. To this mixture was added pentane (160ml), and the precipitated substances were filtered off and washed with pentane. The filtrate was concentrated, to give a residue, which was purified by a silica gel column chromatography (developer: n-hexane) to give 7 (1.2058g, yield 87%). Synthesis Example 7 After a solution of 7 (1.2058g, 3.57mmol) in THF (40ml) was cooled to 0°C under a nitrogen atmosphere, n-butyllithium (hexane solution, 1.70M, 4.0ml) was added with stirring. After the reaction mixture was stirred at -78°C for 1 hour, it was warmed to the room temperature over a period of 1 hour and stirred at the room temperature for further 1 hour. Thereafter it was cooled again to -78°C, veratoraldehyde (0.622g, 3.75mmol) was added and then warmed to the room temperature for 2 hours, and then stirred at the room temperature overnight. An aqueous ammonium chloride solution was added and the mixture extracted with ethyl acetate. After the extract was dried over sodium sulfate, the solvent was distilled off. A residue was purified by a silica gel column chromatography (developer: n-hexane:ethyl acetate=5:1) to give 8 (0.99g, yield 81%). Example 1 Synthesis of compound No. 6 in Table-1 A solution of aminoalcohol (1.20g, 3.93mmol) obtained in Synthesis Example 2 in dichloromethane (50ml) was stirred vigorously at the room temperature, and active manganese dioxide (12.0g) was added to the solution. After 1 hour, insolubles were filtered off, the filtrate was concentrated under reduced pressure, and the residue was purified by neutral alumina column chromatography (developer: n-hexane/ethyl acetate=4/1) to afford the above-noted objective ynone compound (715mg, yield 60 %) as an oil. NMR spectra, form and IR spectra are as follows. Form: brown oil 1 H NMR (250 MHz, CDCl 3 ) δ ppm : 1.16 (d, J = 6.5 Hz, 6H), 3.27 (sep, J = 6.5 Hz, 1H), 3.73 (s, 2H), 3.94 and 3.97 (2s, 6H), 6.94 (d, J = 8.5 Hz, 1H), 7.62 (d, J = 1.8 Hz, 1H), 7.84 (dd, J = 1.8, 8.5 Hz, 1H). IR (film) cm -1 : 2969, 2936, 2209, 1636, 1584, 1514, 1464, 1420, 1343, 1271, 1219, 1175, 1134, 1024, 880, 824, 766, 750, 723. Example 2 Synthesis of compound No. 9 in Table-1 By using piperidine and propargyl bromide as starting materials analogously to Synthesis Examples 1 and 2 as well as Example 1, the corresponding ynone compound was obtained (total yield 7%). A solution of fumaric acid (80mg, 0.69mmol) in acetone (10ml) was added to a solution of the ynone compound (200mg, 0.70mmol) in acetone (5ml) at the room temperature and stirred. After 5 hours, precipitated crystals were filtered off, washed with acetone, and then dried under a decreased pressure to give the above-noted amine fumarate (50mg, yield 18%) as colorless crystals. NMR spectra, melting point and IR spectra are as follows. mp.: 105-106°C 1 H NMR (250MHz, DMSO-d) δ ppm : 1.30-1.70 (m, 6H), 2.51 (m, 4H), 3,67 (s, 2H), 3.84 and 3.88 (2s, 6H), 6. 6 2 (2s, 3 H) , 7.18 (d, J= 8.5 Hz, 1H), 7.52 (d, J=1.8Hz, 1H), 7.79 (dd, J=1.8, 8.5 Hz, 1H). IR (film) cm -1 : 3430, 2938, 2627, 2531, 2236, 1888, 1699, 1638, 1582, 1514, 1456, 1422, 1273, 1219, 1017, 974. Example 3 Synthesis of compound No. 10 in Table-1 By using 2,2,6,6-tetramethylpiperidine as a starting material analogously to Synthesis Examples 1 and 2 as well as Example 1, the above-noted amine compound was obtained (total yield 18 %). NMR spectra, melting point, form and IR spectra are as follows. mp.: 142-144°C Form: light yellow crystals 1 H NMR (250 MHz, CDCl 3 ) δ ppm: 1.16 (s, 12H), 1.30-1.65 (m, 6H), 3,60 (s, 2H), 3. 9 5 and 3. 9 7 (2s, 6H), 6.93 (d, J=8.4Hz, 1H), 7.65 (d, J = 2.0Hz, 1H), 7.85 (dd, J = 2.0. 8. 4Hz, 1H). IR (KBr) cm -1 : 2969, 2934, 2868, 2220, 1630, 1582, 1514, 1458, 1345, 1296, 1271, 1219, 1177, 1132, 1019, 858, 766, 617. Examples 4 ~ 9 By using acetylene compounds (Examples 4 ~ 7) prepared according to the method of A. P. Poisselle et al [J. Org. Chem., 26, 725(1961)] and the method of R. S. Hanze et al [J. Am. Chem. Soc., 82, 4908(1961)] or commercially available acetylene compounds (Examples 8 and 9 ) and veratolaldehyde as starting materials analogously to Synthesis Examples 2 and Example 1, compounds in Example 4 ~ 9 were obtained. Purification in each step was carried out by using a silica gel column chromatography. Structures of acetylene compounds used and objective materials as well as physical property values and yields of objective materials are described as follows. Hydrochloride (Example 5) was obtained by dissolving the ynone compound in ether, adding one equivalent of 4N hydrochloric acid-ethyl acetate solution at an ice-cooled temperature, warming to the room temperature, filtering off and drying under a decreased pressure. Reference Example 1 Synthesis of compound No. 197 A solution of propiolic acid (5.64g, 80mmol) in tetrahydrofuran (100ml) was stirred on an ice-bath, triethylamine (13.4ml, 96mmol), aniline (8.0ml, 88mmol) and phosphorus oxychloride (8.2ml, 88mmol) were added. The reaction mixture was stirred for one night at room temperature, to which water was added with ice cooling. The solvent was distilled off under reduced pressure, and the mixture was extracted with ethyl acetate (50ml×2). The extracts were concentrated and thereafter the residue was purified by a silica gel column chromatography (developer: chloroform/ethyl acetate=2/1) to yield an anilide compound (5.58g, yield 48%). A solution of the anilide compound (492mg, 3.37mmol) in tetrahydrofuran (20ml) was cooled to -70°C, and 2M a solution of litium diisopropyl amide in cyclohexane (3.54ml, 7.08mmol) was added slowly dropwise. After stirring for 1 hour, 3,4-dimethoxy benzaldehyde (560mg, 3.37mmol) was added. The resulting solution was warmed to room temperature for 1 hour, then cooled to 0°C, and thereafter water (10ml) was added. After the solvent was distilled off under reduced pressure, water (30ml) was added to the residue and the product was extracted with ethyl acetate (50ml). After the extract was dried (anhydrous sodium sulfate) and concentrated, the residue obtained was purified by a silica gel column chromatography (developer: chloroform/ethyl acetate=4/1) to obtain an adduct, an alcohol compound (600mg, 57%). The alcohol compound (580mg, 1.86mmol) was subjected to oxidation reaction analogously to Example 1, and the crude product obtained was purified by a silica gel column chromatography (developer: n-hexane/chloroform/ethyl acetate=1/1/1) and washed with ether-hexane and filtered off to give the above-noted objective ketone compound (85 mg, yield 14%). NMR spectra, form, melting point and IR spectra are as follows. Form: light orange powder mp.: 140-142°C 1 H NMR (250 MHz, CDCl 3 ) δ ppm : 3. 93 and 3. 9 7 ( 2 s, 6 H) , 6.75 (d, J=8.5Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.37 (m, 2H), 7.54 (d, J=1.8Hz, 1H), 7.60 (d, J=8.2Hz, 2H), 7.83 (dd, J = 1.9, 8.5Hz, 1H), 8.36 (brs, 1H). IR (KBr) cm -1 : 3324, 1680, 1630, 1599 1584, 1545, 1510, 1462, 1442, 1420 1321, 1271, 1171, 1144, 1020, 874, 760. Example 10 Synthesis of compound No. 15 in Table-1 Molecular sieve 4A (10g) was added to a solution of 3-amino-3-methyl-1-butene (5.0g, 90% in H 2 O, 54mmol) in tetrahydrofuran (50ml) and stirred for 30 minutes under a nitrogen atmosphere. Under a nitrogen atmosphere, molecular sieve was filtered off and the filtrate was cooled with a dry ice-ethanol bath. Then 1. 63M solution of n-butyllithium in hexane (33.0ml, 54.0mmol) was added. After warming the reaction mixture slowly to 0t, it was cooled again with the dry ice-ethanol bath and water was added to stop reaction. After distilling off the solvent under a decreased pressure, water was added and a product was extracted with dichloromethane (70ml×2). The extracts were dried (anhydrous sodium sulfate) and concentrated, and the obtained oily material was purified by a silica gel column chromatography (developer: chloroform/methanol=50/3) to obtain an adduct, aminoalcohol (7.65g, 57%). NMR spectra, form and melting point of the aminoalcohol are as follows. 1 H NMR (CDCl 3 , 250MHz) δ ppm: 1.44 (s, 6H), 1.98 (brs, 3H), 3. 8 9 and 3.90 (2s, 6H), 5.41 (s, 1H), 6.86 (d, J = 8.7 Hz, 1H), 7.03 - 7. 1 0 (m, 2H). Form: light yellow crystal mp.: 85-87°C Active manganese dioxide (10.2g) was added in 10 portions to a solution of the above-noted aminoalcohol (1.65g, 6.59mmol) in dichloromethane (50ml) at the room temperature. The reaction mixture was stirred for 2 minutes and filtered by a pad of celite. After concentration of the filtrate, the residue was dissolved in diethyl ether (30ml). The resulting solution was stirred under a nitrogen atmosphere on an icebath, and a 4N hydrochloric acid-ethyl acetate solution (1.8ml) was dropwise added and stirred at the same temperature for 10 minutes. The precipitated product was filtered off, dried, and suspended in ethyl acetate-diethyl ether (5/1). After stirring for 30 minutes and filtering off, the objective 4-amino-1-(3,4-dimethoxy)phenyl-1-oxo-4-methyl-2-pentyne hydrochloride (970mg, 52%) was obtained. NMR spectra, IR spectra, form and melting point are as follows. 1 H NMR (CDCl 3 , 250 MHz) δ ppm : 1.91 (s, 6H), 3.89 and 3.91 (2s , 6H), 6.87 (d, J = 8.4 Hz, 1H), 7.42(s, 1H), 7.90 (d, J = 8.4 Hz, 1H). IR (KBr) cm -1 : 3424, 2940, 2843, 2234, 1636, 1584, 1514, 1464, 1422, 1302, 1275, 1217, 1175, 1146, 1019, 748 Form: yellow powder mp.: 139-141°C Example 11 Synthesis of compound No. 41 in Table-1 An 10% aqueous sodium carbonate solution (20ml) and dichloromethane (10ml) were added to the intermediate aminoalcohol (738mg, 2.96mmol) synthesized in Example 10, then the mixture was stirred vigorlously with ice cooling, and benzyl chloroformate (0.51ml, 3.55mmol) was added. After the reaction mixture was warmed slowly to the room temperature, a product was extracted with dichloromethane (50ml) and the extract was washed with brine. The extract was dried (anhydrous sodium sulfate) and concentrated, the residue was dissolved in dichloromethane (60ml), and then active manganese dioxide (6g) was added with stirring at the room temperature. After stirring for 10 min. the mixture was filtered and the filtrate was concentrated. The residue was purified by a silica gel column chromatography (developer: n-hexane/ethyl acetate/chloroform=4/1/1) to give the required carbamate compound (654mg, 58%) as an oil. NMR spectra and IR spectra are as follows. 1 H NMR (CDCl 3 , 250 MHz) δ ppm : 1.72 (s,6 H), 3.94 and 3.95 (2s, 6H), 5.13 (brs, 3H), 6.87 (d, J = 8.5 Hz, 1H), 7.20 - 7.40 (m, 5H), 7.66 (d, J = 1.8Hz, 1H), 7.87 (dd, J = 1.8, 8.5 Hz, 1H). IR (film) cm -1 : 3349, 2982, 2940, 2222, 1725, 1634, 1584, 1514, 1454, 1420, 1345, 1271, 1132, 1076, 1022, 876, 750. Example 12 Synthesis of compound No. 64 in Table-1 An acetylene compound synthesized according to the method by A. P. Poisselle et al [J. Org. Chem., 26, 725(1961)] and the method by R. S. Hanzel et al [J. Am. Chem. Soc., 82, 4908 (1961)], [3-(methylamino)-3-isopropyl-4-methyl-1-pentyne] (1.53g, 10.0mmol), formalin (2ml, 37%) and formic acid (0.75g) were refluxed for 1.5 hours. After the reaction solution was cooled, it was made basic with a 2N-NaOH solution and then extracted with ether (30ml×1, 20ml×1). The extracts were dried (NaSO 4 ) and concentrated under reduced pressure. The residue (920mg) was reacted with veratoraldehyde (850mg, 5.1mmol) analogously to Synthesis Example 2 and Synthesis Example 3 to obtain the above-noted objective inone compound (1.00g, yield 30%) as colorless cubic crystals. mp. 87~88°C 1 H NMR (250MHz, CDCl 3 ) δ ppm : 1.12 and 1.14 (2 d, J = 6.7Hz, 12H), 2.30 (hep, J = 6.7Hz, 2H), 2.53 (s, 6H), 3.94 and 3.97 (2s, 6H), 6.94 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 1.9 Hz, 1H), 7.87 (dd, J = 8.4, 1.9 Hz, 1H), IR (KBr) cm -1 : 2963, 2872, 2830, 2785, 2199, 1628, 1595, 1581, 1512, 1466, 1420, 1267, 1177, 1140, 1088, 1020, 895, 818, 766, 750, 615. Example 13 Synthesis of compound No. 65 in Table-1 Indomethane (1 ml) was added to a solution of the ynone compound (100mg, 0.33mmol) synthesized in Example 5 in toluene, and stirred at 80°C for 6 hours. The solution was cooled to the room temperature, ether (2ml) was added and heated to reflux for 5 minutes. The mixture was cooled to the room temperature and the above-noted objective compound was collected by filtration. mp. 1 H NMR (300 MHz, CDCl 3 ) δ ppm: 1.61 (t, J = 8.2 Hz, 6H), 2. 1 4 (s, 6H), 3.37 (s, 3H), 3.88 (m, 4H), 3. 9 5 and 4. 0 0 (2s, 6H), 7. 0 5 (d, J = 8.4 Hz, 1H) , 7. 5 1 (d, J = 2.0 Hz, 1H), 7.72 (dd, J = 8.4, 2.0Hz, 1H). Example 14 Synthesis of compound No. 53 in Table-1 Disuccinyl carbonate (640mg, 2.5mmol) was added to a solution of 3-pyridine methanol (240ml, 2.5mmol) in dichloromethane (10ml) and the mixture stirred for one night at the room temperature. To this mixture was added a solution of aminoalcohol [4-amino-1-(3,4-dimethoxy)phenyl-4-methyl-2-pentyn-1-ol] (500mg, 2.0mmol) in dichloromethane (5ml) and stirred overnight. Water was added to the reaction mixture and the product was extracted with ethyl acetate. The extract was dried (Na 2 SO 4 ) and concentrated, and the residue was purified by a silica gel column chromatography (developer: chloroform/methanol=50/1) to give a pyridylmethyl carbamate compound (650mg, 67%). 1 H NMR of the pyridylmethyl carbamate compound is as follows. 1 H NMR (250MHz, CDCl 3 ) δ ppm : 1.62 (s, 6H), 3.86 (2s, 6H), 5.06 (s, 2H), 5.43 (s, 1H), 5.48 (b r s, 1H), 6.81 (d, J = 8.3 Hz, 1H), 7.07 (d d. J = 8.3, 1.8 Hz, 1H), 7.13 (d, J = 1.8Hz, 1H), 7.23 (dd, J = 4.7, 7.8 Hz, 1H), 7.63 (dt, J = 7.8, 1.9 Hz, 1H), 8.47 (dd, J = 4.7, 1.5Hz, 1H), 8.55 (m, 1H). The above-noted pyridylmethyl carbamate compound was oxidized analogously to the method of Synthesis Example 3 to obtain an ynone compound (yield 82%) as a brown oil. 1 H NMR (250MHz, DMSO-d+1 drop D 2 O) δ ppm : 1.63 (s, 6H), 3.84 and 3.90 (2s, 6H), 5.16 (s, 2H), 7.09 (d, J = 8.5 Hz, 1H), 7.43 (m, 1H), 7.55 (d, J = 1.6 Hz, 1H), 7.77 (dd, J = 8.5, 1.6 Hz, 1H), 7.83 (d, J = 7.9 Hz , 1H), 8.20 (s, 1H), 8.53 (brd, J = 4.1 Hz), 8.61 (brs, 1H). IR (film) cm -1 : 3345, 3216, 2984, 2220, 1725, 1634, 1583, 1515, 1464, 1422, 1345, 1271, 1082, 1022, 876, 752. Example 15 Synthesis of compound No. 55 in Table-1 Magnesium monoperoxyphthalate (240mg) was added to a solution of the ynone compound (189mg, 0.49mmol) in dichloromethane (5ml) with ice cooling and stirred for 2 hours at the room temperature. Again, magnesium monoperoxyphthalate (240mg) and ethanol (5ml) were added with ice cooling and stirred for 2 days at room temperature. Water was added to the reaction mixture and a product was extracted with dichloromethane (15ml×2), and the extract was washed with a saturated aqueous sodium hydrogensulfite solution and dried (Na 2 SO 4 ). The solvent was distilled off under reduced pressure to give the objective N-oxide compound (178mg, 92%) as light yellow crystals. mp. 65~80°C (not show clear melting point) 1 H NMR (250 MHz, DMSO-d+1 drop D 2 O) δ ppm : 1.63 (s, 6H), 3.84 and 3.89 (2s,6H), 5.10 (s, 2H), 7.11 (d, J=8.5Hz, 1H), 7.45 (m, 2H), 7.54 (d, J=1.6 Hz, 1H), 7.77 (dd, J=1.6, 8.5Hz, 1H), 8.20 (m, 1H), 8.31 (brs, 1H). IR (KBr) cm -1 : 3347, 2982, 2940, 2361, 2220, 1724, 1636, 1584, 1514, 1271, 1171, 1086, 1020, 752. Example 16 Synthesis of compound No. 54 in Table-1 Methyl iodide (0.5ml) was added to a solution of the inone compound (100mg, 0.26mmol) synthesized in Example 14 in ether (5ml) and stirred for 3 days. The precipitates were collected by filtration and dried under reduced pressure to give the objective pyridinium compound (70mg, 51%) as light yellow powdery crystals. mp. 196~197.5°C 1 H NMR (250 MHz, DMSO - d + 1 drop D 2 O) δ ppm : 1.64 (s,6H), 3.84 and 3.88 (2s, 6H), 4.34 (s,3H), 5.20 and 5.35 (2d, J = 36.0 Hz, 2H), 7.13 (d, J=8.5Hz, 1H), 7.54 (s,1H), 7.78 (s, J=8.2 Hz, 1H), 8.11 (m,1H), 8.55 (d,J=8.0Hz, 1H), 8.90 (d, J=6.0 Hz, 1H), 9.00 (s, 1H). Example 17 Synthesis of compound No. 14 in Table-1 A solution of propargylamine (1.65g, 30mmol) in 1N NaOH was stirred on an ice-bath, and di-t-butyl carbonate (6.9ml, 30.0mmol) was added. It was warmed to the room temperature and stirred overnight. It was extracted with ether, and the extract was washed and dried (Na 2 SO 4 ). The solvent was distilled off under a decreased pressure to give a carbamate compound (3.57g, 77%). The obtained carbamate compound was oxidized by the method analogously to Reference Example 1, and reacted with veratrol, and thereafter purified by a silica gel column chromatography (developer: n-hexane/ethyl acetate=1/1) to afford the objective ynone compound (yield 45%) as light yellow crystals. mp. 110~113°C 1 H NMR (250MHz, CDCl 3 ) δ ppm : 1.48 (s, 9H), 3.94 and 3.97 (2s, 6H), 4.22 (d, J=5.6 Hz, 2H), 4.94 (brs, 1H), 6.91 (d, J = 8.5 Hz, 1H), 7.59 (d, J=1.8 Hz, 1H), 7.83 (dd, J=1.8, 8.5 Hz, 1H). IR (KBr) cm -1 : 3351, 3007, 2940, 2836, 2234, 1682, 1620, 1597, 1581, 1508, 1454, 1416, 1354, 1267, 1028, 866, 833, 766. Example 18 Synthesis of compound No. 45 in Table-1 Aminoalcohol (3.34g, 13.4mmol) synthesized in Example 10 was reacted with ethyl chloroformate (1.54ml, 16.1mmol) by the method analogously to Example 11, and thereafter oxidized with manganese dioxide (12.2g). The crude product was purified by a silica gel column chromatography (developer: n-hexane/ethyl acetate=2/1) to give the objective title compound (4.27g, quantative) as colorless cubic crystals. mp. 72.0~73.5°C 1 H NMR (250 MHz, CDCl 3 ) δ ppm: 1.26 (t, J=7.1 Hz, 3H), 1.72 (s, 6H), 3.97 (s, 6H), 4. 1 4 (q, J=7.1 Hz, 2H), 4. 9 6 (brs, 1H), 6.93 (d, J = 8.5Hz, 1H), 7.68 (d, J = 1.9 Hz, 1H), 7. 9 0 (dd, J = 8.5, 1.9 Hz, 1H). IR (KBr) cm -1 : 3329, 2982, 2228, 1709, 1626, 1591, 1512, 1464, 1421, 1211, 1173, 1146, 1084, 1020, 756. Example 19 Synthesis of compound No. 56 in Table-1 By using aminoalcohol synthesized in Example 10 and 3,4-dimethoxy sulfonyl chloride in the method analogously to Example 11, sulfonamidation and oxidation were carried out to convert to an ynone compound. The crude product was purified by a silica gel column chromatography (developer: chloroform/methanol=75/1) to give the titled compound as a brown oil (yield 13%). 1 H NMR (250 MHz, CDCl 3 ) δ ppm : 1.67 (s, 6H), 3.80 and 3.84 (2s, 6H), 3.95 and 3.97 (2s, 6H), 5.06 (brs, 1H), 6.79 (d, J=8.6 Hz, 1H), 6.91 (d, J=8.4 Hz, 1H), 7.36 (d, J=2.2 Hz, 1H), 7.54 (dd, J=2.2 Hz, 8.6 Hz, 1H), 7.57 (d, J=1.9 Hz, 1H), 7.68 (dd, J=1.9, 8.4Hz, 1H). IR (film) cm -1 : 3266, 3021, 2940, 2841, 2220, 1636, 1589, 1512, 1464, 1271, 1140, 1022, 754. Example 20 Synthesis of hydrochloride free compound No. 5 in Table-1 The starting material, 3-diethylamino-1-propyne, was prepared according to Synthesis Example 1, from which the above-noted ynone compound (free hydrochloride) was synthesized (yield 47%). 3.0g (10.89mmol) of the above-noted ynone compound (free hydrochloride) was dissolved in 200ml of methylene chloride, and 5.8ml of a solut of 4N-hydrochloric acid-ethyl acetate was added dropwise under a nitrogen atmosphere at 0°C. It was stirred for 20 minutes, then filtered, and dried under reduced pressure to give 2.9g (85%) of the above-noted hydrochloride of the inone compound. m.p. 140-142°C 1 H NMR (250MHz, CDCl 2 ) δ ppm : 1.55 (t, 6H, J=7.3 Hz), 3.08-3.23 (m, 4H), 3.96 (s, 3H), 3.99 (s, 3H), 4.23 (d, 2H, J=2.1 Hz), 6.98 (d. 1H, J=8.4 Hz), 7.55 (d. 1H, J = 2.0Hz), 7.86 (dd, 1H, J=8.3.2.0Hz), 13.22 (brs, 1H). IR (film) cm -1 : 3426, 2946, 2922, 2637, 2556, 2486, 2228, 1638, 1580, 1516, 1470, 1449, 1343, 1298, 1277, 1219, 1138, 1017, 860, 829. Examples 21-36 By using various acetylene compounds synthesized according to the method by A. P. Poisselle et al [J. Org. Chem., 26, 725(1961)] and the method by R. S. Hanzel et al [J. Am. Chem. Soc., 82, 4908(1960)] and various aldehydes as starting materials in the methods analogously to Synthesis Example 2 and Example 1, various ynone compounds were synthesized. Purification in each step was carried out by using a silica gel column chromatography. Hydrochlorides were obtained by adding one equivalent of a solut of hydrochloric acid-ethyl acetate warming to the room temperature, filtering off and drying under reduced pressure. Starting materials used as well as physical properties and the yield of the objective materials are described as follows. Example 37 Synthesis of hydrochloride of compound No. 57 in Table-1 Under a nitrogen atmosphere 2.8ml of n-butyllithium (1.6M in hexane) was added dropwise to a solution of the above-noted amine (1) (0.38g, 2.25mmol) in anhydrous tetrahydrofuran at -78°C (20ml) and the resulting solution was stirred for 1 hour. To this solution was added a solution of veratoraldehyde (2) (0.37g, 2.25mmol) in anhydrous tetrahydrofuran (5ml) and the solution was stirred for 1 hour. Water was added to the solution and the solvent was distilled off under reduced pressure. After the residue was extracted with 2N HCl aqueous solution and the aqueous layer was washed with ethyl acetate, 2N NaOH a solution was added to the aqueous layer to make pH above 13, and the mixture extracted with dichloromethane. After drying organic layer over sodium sulfate, the solvent was distilled off under reduced presure. The residue obtained was washed by a silica gel column chromatography (ethyl acetate → chloroform:methanol=8:1) to obtain 0.46g of alcohol compound (3) (yield 60%). Manganese dioxide (200mg) was added to a solution of the alcohol compound (3) (36mg, 0.10mmol) in anhydrous dichloromethane (5ml) and stirred at the room temperature. After insolubles were filtered off and 25µl of 4N HCl-ethyl acetate solution was added to a filtrate, the solvent was distilled off to obtain 10mg (yield 27%) of (4) as hygroscopic crystals. 1 H NMR (CDCl 3 , 250 MHz) δ ppm : 1.21 (3H, d, J=6.6Hz), 1.27 (3H, d, J=6.6 Hz), 1.79 (3H, s), 1.96~2.13 (2H, m), 2.27~2.45 (1H, m), 2.50~2.69 (1H, m), 3.42~3.65 (2H, m), 3.83~3.98 (1H, m), 3.95 (3H, s), 3.98 (3H, s), 4.05~4.29 (1H, m), 7.05 (1H, d, J=8.5 Hz), 7.57 (1H, d, J=1.9 Hz), 7.95 (1H, dd, J=8.5, 1.9 Hz), 9.24 (1H, brs), 10.60 (1H, brs). Example 38 Synthesis of hydrochloride of compound No. 58 in Table-1 35% aqueous formaldehyde solution was added to a solution of the alcohol compound (3) (150mg, 0.45mmol) synthesized in Example 61 in acetonitrile (0.2ml, 2.3mmol), then 71mg (0.90mmol) of NaBH 3 CN was added and stirred for 2 hours at room temperature. After 3ml of glacial acetic acid was added dropwise, the reaction mixture was concentrated under reduced pressure, to which 4N KOH aqueous solution was added to make basic, and then extracted with ether. After the organic layer was extracted with 1N HCl aqueous solution, an aqueous layer was made basic with 4N KOH aqueous solution, and extracted with ether. After the organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by a silica gel column chromatography (chloroform:methanol=20:1) to give 98mg (yield 62%) of (5). 350mg of manganese dioxide gas was added to a solution of (5) (98mg) in dichloromethane (5ml) and stirred for 1 hour at room temperature, and thereafter insolubles were filtered off and 0.12ml of 4N HCl-ethyl acetate solution to a residue, then the solvent was distilled off to obtain 45mg (yield 41%) of (6). 1 H NMR (CDCl 3 , 300 MHz) δ ppm : 1.35 (6H, d, J=6.6 Hz), 1.83 (3H, s), 1.93~2.20 (4H, m), 2.40~2.60 (2H, m), 3.07 (3H, s), 3.1 2~3.31 (1H, m), 3.67~3.83 (2H, m), 3.89 (3H, s), 3.99 (3H, s), 7.00 (1H, d, J=8.4 Hz), 7.54 (1H, d, J=2.1 Hz), 7.88 (1H, dd, J=8.4, 2.1 Hz), 11.40 (1H, brs). Test Example 1 <Evaluation of tyrosine kinase inhibitor of the present invention> In order to evaluate tyrosine kinase inhibitory activity and cancer cell growth inhibitory activity for tyrosine kinase inhibitor of the invention, tests were carried out in partially purified human EGF (epidermal growth factor) receptor tyrosine kinase activity detemination system and in a cell incubation system using human cancer cells. Furthermore, in order to compare and evaluate strength of inhibitory activity, relatively higher active ones amongst known tyrosine kinase inhibitors disclosed in patents or literatures were tested simultaneously. (1) Tyrosine kinase inhibitory activity (Determination method) Tyrosine kinase activity was measured using partially purified EGF receptor prepared from A431 cell line derived from human epidermoid carcinoma according to the improved method of that of Linda J. Pike et al (Proceedings of the National Academy of Sciences of the U.S.A., 79 1443, 1982). The method employed is detailed below. A431 cells were cultured in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal calf serum (FCS) at 37°C under 5% CO 2 atmosphere. The cultured cells were homogenated in a solution containing 10mM N-2-hydroxyethylpiperazino-N'-2-ethane sulfonate buffer (pH 7.4), 0.25M sucrose, and 0.1mM EDTA, and centrifuged at 3000×g for 5 minutes. The supernatant was then centrifuged at 10000×g for 30 minutes to obtain A431 cell membrane fraction. The cell membrane fraction, a partially purified EGF receptor, was used as an enzyme source. A test compound dissolved in dimethylsulfoxide (DMSO) was added to a reaction mixture containing 15mM Hepes buffer (pH 7.7), 2mM MnCl 2 , 10µM ZnSO 4 , 50µM Na 3 VO 4 and the partially purified EGF receptor preparation (10-15 µg) (final concentration 1% DMSO). To this mixture 100ng EGF, 75µg synthetic substrate RR-SRC peptide shown by Sequence No. 1 in Sequence table and 10µM γ- 32 P-adenosine triphosphate (55.5 KBq) were added thereto to start the reaction. The volume at that time was 60 µl. RR-SRC peptide is a substrate for tyrosine kinase of EGF receptor and has an amino acid sequence containing tyrosine residue which is phosphorated in a gene product of src. The mixture was allowed to react under ice-cold for 30 minutes, and the reaction was stopped by addition of 10 mg/ml bovine serum albumin and 20% trichloroacetic acid. The reaction mixture was left to stand under ice-cold for 30 minutes. The mixture was centrifuged at 5000×g for two minutes, and a 40 µl aliquot of the supernatant was adsorbed on P81 phosphocellulose paper. The paper was dipped in 30% aqueous acetic acid for 15 minutes for fixation and then washed with 15% acetic acid. The washing was repeated four times. The radioactivity of 32 P retained on the phosphocellulose paper was counted with liquid scintillation counter. The value is A. Count numbers in the reaction without addition of test material as well as in the reaction without addition of test material and EGF were determined simultaneously, those values being B and C respectively. Tyrosine kinase inhibitory ratio can be calculated from those values according to the following formula: Inhibitory rate (%) = {(B-A)/(B-C)} × 100 The value IC 50 value (concentration of a test compound providing 50% inhibition) was calculated from inhibitory rate at various concentrations of a test compound in the above-described test procedure. Cancer Cell Growth Inhibitory Activity KB cell, which is a human rhinopharyngeal cancer cell, possesses overexpression of EGF receptors. Accordingly, the KB cell was used to evaluate the effect of test compounds on the growth of cultured cancer cells. KB cells were plated on a 96-well dish (2.5×10 3 cell/well) and cultured for 24 hours at 37°C under 5% CO 2 atmosphere in DMEM:F12 (1:1) medium containing 10% FCS, 50 µg/ml penicillin, and 50µg/ml streptomycin. To the culture medium was added a test compound dissolved in DMSO (DMSO final concentration < 0.1%), and the cells were cultured for three days under the same conditions. The test compound was exchanged every 24 hours together with the culture medium. The amount of living cells were measured by calorimetric determination at wave lengths of 550 nm and 650 nm after developing colour by MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), in accordance with the method of Michael C. Alley et al. (Cancer Research 48 589, 1988). Cancer cell growth inhibitory activity (CCGIA) was determined according to the following equation. CCGIA (%) = (b-a)/b × 100 Symbols "a" and "b" represent the colorimetric values determined in the cancer cell growth assay with and without the test compounds, respectively. The value IC 50 (concentration of a test compound providing 50% inhibition) was calculated from CCGIA values at various concentration of a test compound in the above-described procedure. Results are shown in Table-2 and Table-3, wherein "Compound No." in the first column corresponds to that in Table-1. 1) A.Gazit, N.Osheroy, I.Posner, P.Yaish, E.Pradosu, C.Gilon and A.Levitzki; J. Med. Chem., 34, 1896(1991). 2) T.Shiraishi, K.Kameyama, N.Imai, T.Domoto, I.Katsumi and K.Watanabe; Chem. Pharm. Bull., 36, (1988). 3) fumarate, 4) hydrochloride From the above-noted results, it is clear that the compounds according to the invention are superior in tyrosine kinase inhibiting activity and cancer cell growth inhibiting activity. [Sequence Table] Sequence number: 1 Length of sequence: 13 Type of sequence: amino acid Topology: straight chain Kind of sequence: peptide Sequence

Claims: A compound of the following formula (I) wherein R 2 and R 3 are C 1 -C 3 alkoxyl R 6 is phenyl, pyridyl or -CR 15 R 16 X wherein R 15 and R 16 are independently hydrogen or C 1 -C 5 alkyl. optionally substituted by phenyl, or when taken together, they are C 3 -C 7 alkylene, X is hydroxyl or -NR 23 R 24 wherein R 23 and R 24 are independently hydrogen, phenyl optionally substituted by halogen or C 1 -C 5 alkyl, C 1 -C 5 alkyl optionally substituted by hydroxyl, phenyl or indolyl, pyridyl, -SO 2 R 27 wherein R 27 is 3,4-dimethoxyphenyl, or -COR 25 wherein R 25 is C 1 -C 5 alkoxy optionally substituted by phenyl, pyridyl, N-methylpydridyl or N-oxopyridyl, or when R 23 and R 24 are taken together, they are C 3 -C 6 alkylene optionally substituted by C 1 -C 5 alkyl; or its salt, except 2-propyn-1-one, 1-(3,4-dimethoxyphenyl)-3-phenyl. A tyrosine kinase inhibitor. which comprises as an essential ingredient a benzoylacetylene derivative of formula (I) as defined in claim 1 or its salt. A pharmaceutical composition which comprises a compound of claim 1 and pharmaceutically acceptable carriers. The composition of claim 3 for suppressing the growth of cancer cells. The composition of claim 4, wherein the cancer cell is of stomach cancer, breast cancer, ovary cancer, lung cancer, colon cancer. brain cancer, leukemia, pancreatic cancer or esophageal cancer. The composition of claim 3 for therapeutical treatment of arterial sclerosis. The composition of claim 3 for inhibiting platelet aggregation. The composition of claim 3 for immunodepression. The composition of claim 3 for suppressing inflammation.

Inventors: INOKAWA HARUKI, KITANO YASUNORI, SUZUKI TSUYOSHI, TAKAYANAGI HISAO, INOKAWA, HARUKI, KITANO, YASUNORI, SUZUKI, TSUYOSHI, TAKAYANAGI, HISAO
Applicants: MITSUBISHI CHEM CORP, MITSUBISHI CHEMICAL CORPORATION
Application year: 1994
Main classification: C07D295
IPC codes: C07D213, C07C69, C07C45, C07D317, C07C43, C07D295, C07C49, C07C225, C07C205, C07C311, C07C275, C07F7, C07C235, C07C17, C07C271, C07D209